HIV latency represents a key barrier preventing the cure of infected individuals through the use of antiretroviral therapy (ART) alone. Historically, efforts to deplete latently-infected cells have been hampered by a lack of relevant small animal models that can be used to study HIV latency and evaluate methods for eliminating latent virus. To address this issue we recently developed a new in vivo model for HIV latency using the humanized BLT (bone marrow-liver-thymus) mouse. We demonstrated that HIV forms latency within infected BLT mice, and that the latent virus is integrated, activation-inducible, replicatio-competent, and responds to known HIV latency reversing agents (LRAs). This model represents a versatile tool for studying latent HIV. However, challenges still exist within the HIV latency field, particularly in accurately quantifying latently-infected cells and in evaluating activation-elimination approaches that cause significant but incomplete depletion of persistent/latent reservoirs of HIV. Here, we propose to develop a new system for analyzing HIV persistence and latency using genetically barcoded virus. We have created an HIV swarm composed of >200,000 different viral variants that are genetically identical except for the presence of a short non-expressed, phenotypically neutral barcode sequence. This barcoded virus replicates efficiently in vitro, is pathogenic in vivo in BLT mice, and has multiple potential applications in HIV cure research. We intend to further develop and test the barcoded virus in the context of the humanized BLT mouse latency model. We will use deep sequencing approaches to evaluate viral diversity following ex vivo stimulations of latently-infected cells from bulk tissue samples, and thereby compare the relative efficacy of different individual or combination LRAs. We will also test our hypothesis that barcoded virus will allow us to monitor the diversity of HIV plasma virus that emerges from reservoirs upon cessation of ART. This would set the stage for in vivo testing of LRAs, with reductions in barcode diversity serving as a new additional measure of the effectiveness of LRAs in a whole animal system. Together, these experiments will provide important new information on HIV latency and will form the basis of future in vivo modelling of HIV cure approaches.